Thyroid status in juvenile alligators (Alligator

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Chemosphere 47 (2002) 1129–1135
www.elsevier.com/locate/chemosphere
Thyroid status in juvenile alligators
(Alligator mississippiensis) from contaminated and reference
sites on Lake Okeechobee, Florida, USA
Elizabeth A. Hewitt a, D. Andrew Crain a,*, Mark P. Gunderson b,
Louis J. Guillette Jr b
a
Department of Biology, Maryville College, 502 Lamar Alexander Parkway, Maryville, TN 37804, USA
b
Department of Zoology, 223 Bartram Hall, University of Florida, Gainesville, FL 32611, USA
Received 14 August 2000; accepted 8 February 2002
Abstract
Exposure to environmental contaminants has been shown to alter normal thyroid function in various wildlife
species, including the American alligator (Alligator mississippiensis). Abnormalities in circulating levels of the thyroid
hormone thyroxine (T4 ) have been reported in juvenile alligators from several contaminated lakes in Florida. To further
elucidate these functional thyroid abnormalities, this study examines the structure of thyroids and circulating T4
concentrations from juvenile alligators collected from three sites of varying contamination on Lake Okeechobee,
Florida. The following variables were used to characterize thyroid morphology: epithelial cell height, width and area,
percent colloid, and follicle area. These variables were compared among study sites and between genders. No difference
was detected in epithelial cell height, epithelial cell area, or follicle area among the sites, whereas significant differences
in epithelial cell width (p ¼ 0:02) and percent colloid (p ¼ 0:008) were found. Animals from the most contaminated site
(Belle Glade) had significantly greater epithelial cell widths and significantly less colloid present in their follicles
compared to animals from the reference site (West). Gender did not have a significant interaction with site for any
variable measured. Thyroxine (T4 ) concentrations were elevated in the intermediately contaminated site (Conservation
Area 3A) compared to the other sites (p < 0:0001). It is proposed that the disruptions seen in Lake Okeechobee alligators are due to disruptions at both the thyroid and extra-thyroidal tissues. Ó 2002 Elsevier Science Ltd. All rights
reserved.
Keywords: Alligator; Endocrine disruption; Contaminants; Thyroid gland, thyroxine
1. Introduction
The thyroid is the endocrine gland responsible for the
synthesis and secretion of the hormones thyroxine (T4 )
and triiodothyronine (T3 ). Thyroid hormones are essential for the regulation of several major functions in all
*
Corresponding author. Tel.: +1-865-981-8238; fax: +1-865981-8010.
E-mail address: [email protected] (D.A. Crain).
vertebrates, including metabolism and growth and
development of body systems; other specific functions
include regulating metamorphosis in amphibians and
influencing reproduction (Schmidt-Nielsen, 1990; Dellovade et al., 1995). In light of the various roles of
thyroid hormones, the consequences of thyroid disruption can be severe. Embryonic effects of thyroid deficiency include limited development of skeletal, muscle,
and central nervous systems (McNabb and King, 1993).
Several recent studies have noted altered thyroid activity as a result of exposure to environmental chemicals,
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 5 - 6 5 3 5 ( 0 2 ) 0 0 0 9 0 - 5
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E.A. Hewitt et al. / Chemosphere 47 (2002) 1129–1135
specifically endocrine-disrupting contaminants (EDCs).
Although some of the most profound effects of EDCs
are elicited in the reproductive glands of the endocrine
system, thyroid gland disruption was one of the first
observed effects of these chemicals and has since been
well documented (see reviews in Leatherland (1992,
2000)). Elevated circulating T4 levels coupled with depressed T3 levels are noted in freshwater catfish (Clarias
batrachus) experimentally exposed to the pesticide endosulfan (an organochlorine), suggesting that this pesticide blocks extrathyroidal conversion of T4 to T3
(Sinha et al., 1991). Unlike endosulfan, carbaryl (a carbamate) caused a decrease in T4 levels and an increase in
T3 levels in C. batrachus (Sinha et al., 1991). A similar
correlation between contaminant exposure and thyroid
dysfunction has been noted in rats. Extreme hypothyroidism in weanling male rats was provoked by administration of the PCB mixture Aroclor 1254 (Gray et al.,
1993).
In addition to functional abnormalities, numerous
studies have noted structural abnormalities in the thyroids of wildlife exposed to environmental contamination. The constituents of thyroid follicles (epithelial cells,
colloid, and individual follicles themselves) are instrumental in observing structural effects of EDCs on the
thyroid. Histologically, epithelial cell height is the
most frequently used method of thyroid gland assessment, as it is considered to be roughly proportional to
the degree of response to thyroid-stimulating hormone,
TSH (Moccia et al., 1981). Also useful are epithelial cell
width, colloid content, and follicle diameter. Normal
thyroid morphology––smaller follicles with columnar
epithelial cells and abundant colloid––may be altered
with prolonged exposure to contamination. Large follicles with cuboidal or flattened epithelial cells and severely depleted colloid have been observed in herring
gulls from the Great Lakes basin, an area exhibiting
prevalent thyroid disorder in fish, avian, and mammal
species (Moccia et al., 1986; Leatherland, 1994). These
structural abnormalities noted in Great Lakes species
are thought to have an environmental etiology (Moccia
et al., 1981).
Recently, thyroidal abnormalities have been noted
in American alligators (Alligator mississippiensis). This
species, as for reptiles in general, is highly susceptible
to endocrine disruption by contamination (Crain and
Guillette, 1998). Reproductive abnormalities persisting
in the juvenile alligator population of Lake Apopka
(Florida), site of a major pesticide spill in 1980, include
altered sex-steroid concentrations, altered gonadal
morphology, and reduced phallus size. Juvenile alligators collected from Lake Okeechobee, Florida (a site of
significant agricultural runoff) have similar, although
less pronounced, abnormalities in sex-steroid concentrations (Crain et al., 1998). In addition to reproductive
abnormalities, altered thyroid hormone concentrations
have been observed in Lake Okeechobee juvenile alligators (Crain et al., 1998). Circulating levels of thyroxine were significantly higher in Okeechobee male
alligators compared to control animals. The present
study seeks to examine both thyroid structure and thyroid function in Okeechobee juvenile alligators in order
to elucidate previously reported functional abnormalities in this population. It is hypothesized that thyroid
morphology and thyroxine concentrations will differ in
animals from contaminated and reference sites.
2. Materials and methods
2.1. Study sites
Lake Okeechobee (lat. 26°560 N, long. 80°490 W), is
the largest lake in Florida and was chosen for this study
based on its historical exposure to environmental contamination as well as the previously reported alterations
in thyroid status of its alligators. In the South Florida
Water Management District (SFWMD), which is responsible for water-resource management in South
Florida, the total estimated annual usage of pesticides as
of 1996 was 14 590 tons/year (Miles and Pfeuffer, 1997).
Pesticides including insecticides, herbicides, and fungicides are used in this area primarily for agriculture
(sugarcane, citrus, and vegetable crops). Other uses include golf course maintenance, domestic uses, and mosquito control (Miles and Pfeuffer, 1997).
Animals were collected from three sites on or near
Lake Okeechobee: Belle Glade (May 11, 1999), West
(May 10, 1999), and Conservation Area 3A North (May
12, 1999). Belle Glade is located in the Southeastern
portion of the lake and is adjacent to major sugarcane
agriculture. In fact, 100% of Florida’s sugarcane industry is located on the Southern-most edge of Lake Okeechobee (see Fig. 1). The herbicides ametryn, atrazine,
and 2,4-D are predominantly used on sugarcane (Miles
and Pfeuffer, 1997). West, an area considered to be relatively pristine, is located in the western-most part of the
lake, an area that has little agriculture (see Fig. 1).
Conservation Area 3A is the largest of five Conservation
Areas located South of Lake Okeechobee and North of
the Everglades National Park (see Fig. 1). These Conservation Areas, created in the 1950s and 1960s for
water-management purposes, are shallow wetlands enclosed by levees. Conservation Area 3A releases water
into the major undisturbed part of the Everglades
(Mattraw et al., 1988). Water inflow and outflow
structures located throughout the Conservation Areas
represent major links in the water conveyance system
from Lake Okeechobee and the Everglades Agricultural
Area to the Everglades National Park (Lutz, 1977;
Mattraw et al., 1988). Animals were collected in the
Northern part of area 3A near inflow structure S-8.
E.A. Hewitt et al. / Chemosphere 47 (2002) 1129–1135
1131
were found at detectable levels in water or sediment
samples obtained from station FECSR78 at the West
site; and four compounds were found at detectable levels
in samples from pump station S-8 in Conservation Area
3A North (Pfeuffer, 1991; see Table 1). These data indicate that Belle Glade and surrounding areas have been
sites of persistent agricultural runoff and environmental
contamination, whereas detectable levels of chemicals in
the West location have seldom been found. Conservation Area 3A North appears to be an area of moderate
contamination.
2.2. Animal collection and sample processing
Fig. 1. A Map of South Central Florida. The three study sites
(West, Belle Glade, and Conservation Area 3A North) are
shown with their position relative to different agriculture
practices (adapted from Miles and Pfeuffer (1997, p. 338)).
Pump station S-8 is adjacent to the Everglades Agricultural Area and is subject to extensive agricultural
usage, primarily sugarcane (see Fig. 1).
Historically, the three sites have been differentially
exposed to agricultural toxicants. In a pesticide residue
study conducted from 1984 to 1988 within the SFWMD,
chemicals (especially DDT metabolites) were consistently found in water and sediment samples obtained at
pump station S-2 at the Belle Glade site; no compounds
Juvenile alligators were hand-caught from an airboat
over a period of three nights. Immediately upon capture,
a 3 ml blood sample was collected in a vacutainer treated
with sodium heparin. Samples were stored on ice for 6–9
h before centrifugation. Plasma was stored at 72 °C
prior to radioimmunoassay for T4 , which was conducted
as previously described (Crain et al., 1998). Briefly, 100
ll of unextracted plasma was incubated with T4 antibody (1:2000 final concentration; Endocrine Sciences,
Calabasas Hills, CA) and T4 radiolabel (50 000 cpm/
tube; New England Nuclear) in a 0.5 M borate buffer
supplemented with 1% BSA, 1.25 mg/ml c-globulin, and
2 mg/ml 8-alimino-1-naphthalene-sulfonic acid. Hundred ll of plasma stripped of T4 was added to each
standard tube. Tubes were incubated at 37 °C for 2 h,
followed by room temperature incubation for 1.5 h.
Bound-free separation was accomplished by adding 1.5
ml of 60% saturated ammonium sulfate to each tube,
vortexing, and centrifuging at 1500 g for 30 min. The
pellet was resuspended in a 9:11 mixture of saturated
ammonium sulfate and assay buffer with 0.5% bovine
serum albumin. After vortexing and centrifugation, the
pellet was counted on a Beckman gamma counter.
After blood was collected from the alligators, weight,
gender, and size (snout-vent length and total length)
were determined for each animal. Approximately 10
animals (5 males and 5 females) were kept from each
site. Animals were tagged and taken to the field station
where they were euthanized by administering a lethal
injection of sodium pentobarbital into the postcranial
sinus. The thyroid gland was removed and immediately
placed in Bouin’s fixative. Once the fixative had permeated, the preserved tissues were transferred to 75%
alcohol for clearing.
A histological analysis of the juvenile thyroids was
performed in order to study the structure of the glands.
The tissues remained in a solution of 75% alcohol prior
to histological processing. Due to the large size of
juvenile alligator thyroids, the tissues were cut in half
using a scalpel. Tissues were embedded in paraffin wax,
sectioned at 7 lm, and stained with a modified Harris’
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E.A. Hewitt et al. / Chemosphere 47 (2002) 1129–1135
Table 1
Environmental chemicals found at detectable levels in water and sediment samples on Lake Okeechobee and surrounding Conservation Areas (data compiled from Pfeuffer (1991))
Compound
Date
Belle Glade
Conservation Area 3A North
West
Zinc phosphide
Atrazine
p,p0 DDD
p,p0 DDE
June 1986
May 1987
Feb. 1988
Oct. 1987
Feb. 1988
July 1988
Feb. 1986
July 1987
Feb. 1986
6 lg/l
0.4 lg/l
7.9 lg/kg
59.6 lg/kg
10.0 lg/kg
28.0 lg/kg
1100 lg/kg
98.5 lg/kg
3200 lg/kg
3 lg/l
0.3 lg/l
0
0
0
0
1100 lg/kg
0
3300 lg/kg
NS
0
0
0
0
0
0
0
NS
Diazininon
Ametryn
Malathion
trichrome procedure as previously described (Presnell
and Schreibman, 1997).
2.3. Histological analyses
In order to characterize the structure of the thyroid
glands of juvenile alligators, several indices were measured including follicle height and width, epithelial cell
height and width, and percent colloid. Ten follicles
were randomly selected from each tissue. Only spherical follicles that were complete cross-sections were
measured. All measurements were obtained using a
Nikon Alphaphot-2 YS2 microscope with an ocular
micrometer.
The width and height of a chosen follicle were measured at either 100 or 400 magnification depending
on the size of the follicle. The diameter of each follicle
was determined using the equation for calculating the
diameter of an ellipse, p=4ðABÞ, where A and B represent
height and width. For epithelial cell height, the cell
with the greatest height was measured at a magnification of 400. The same epithelial cell was used to obtain a measurement for epithelial cell width at 1000
magnification. The area of that cell was then determined
using the equation for calculating the area of a rectangle,
lw, where l and w represent height and width, respectively. The amount of colloid present in each follicle was
quantitatively measured using digital image analysis.
For the 10 follicles from each thyroid, a Nikon 990
digital camera captured the image and then the percent colloid was determined by image analysis software provided by the National Institutes of Health
(NIH image).
2.4. Statistical analysis
For each of the variables, an analysis of variance
(ANOVA) was conducted to determine if there were any
differences among sites. First, the interaction of site and
gender was analyzed to determine if gender was a significant variable. If not, gender was removed from the
analysis and a single factor ANOVA was conducted to
determine if the response variables differed among sites.
In order to determine where the variation was found, the
Tukey–Kramer post-hoc method was conducted for
data found to be significantly different by the ANOVA
test.
3. Results
In respect to thyroxine concentrations, there was no
interaction between gender and site (p ¼ 0:90). However
T4 was significantly different among sites (p 5 0:0001),
with animals from Conservation Area having significantly higher T4 concentrations compared to animals
from both Belle Glade (p ¼ 0:0001) and West (<0.0001).
Belle Glade and West were not different from each other
(p ¼ 0:64).
The variables used to characterize thyroid morphology (epithelial cell height, width, and area, percent colloid, and follicle area) were first analyzed to determine if
there was a significant interaction between gender and
site. There was no such significant interaction for any of
these morphological variables (epithelial height p ¼
0:69; epithelial width p ¼ 0:14; epithelial area p ¼ 0:19;
percent colloid p ¼ 0:29; follicle area p ¼ 0:29). Thus,
gender was removed from the analysis and morphological variables were compared among the three sites.
Table 2 presents mean values for the morphological
variables and thyroxine concentrations in animals from
the three study sites. There was no difference in mean
epithelial cell height (p ¼ 0:13), mean epithelial cell area
(p ¼ 0:24), or follicle area (p ¼ 0:46) among the three
sites. Significant differences were found in percent colloid (p ¼ 0:008) and epithelial cell width (p ¼ 0:02)
among study sites. The Tukey–Kramer method revealed
that Belle Glade animals, compared to animals from the
reference site (West), had significantly less colloid present within their thyroid follicles and had significantly
greater epithelial cell width. This gave the cells a more
cuboidal or flattened appearance compared to the columnar shape of normal cells.
E.A. Hewitt et al. / Chemosphere 47 (2002) 1129–1135
1133
Table 2
Mean values (1 SE) for measured variables in alligators from the study sites
Epithelial cell height
(lm)
Epithelial cell width
(lm)
Epithelial cell area
(lm2 )
Percent colloid
Follicle area (lm2 )
Thyroxine (ng/ml)
a;b
Belle Glade
Conservation Area
West
p-Value
12:92 1:08
15:42 0:80
14:68 0:97
0.129
5:44 0:27a
4:86 0:26
4:17 0:39b
0.0234
66:11 3:75
72:46 3:00
60:56 6:53
0.241
80:36 3:19a
8:41 104 3:3 104
3:64 0:32a
89:90 2:61
4:72 104 6:5 103
7:32 0:96b
93:39 2:53b
5:40 104 1:2 104
3:25 0:24a
0.008
0.459
<0.0001
Values with different superscripts are significantly different at p 6 0:05, as indicated by the Tukey–Kramer post hoc method.
4. Discussion
The results of this study indicate that both structural
and functional differences in thyroid status exist among
juvenile alligators collected from different sites on Lake
Okeechobee. Structurally, epithelial cell width and percent colloid were found to be significantly different
among the three study sites. Animals from the most
contaminated site, Belle Glade, showed increased epithelial cell widths and decreased percent colloid compared to animals collected from the reference site, West.
A relationship between gender and thyroid morphology
could not be established in this study. Functionally,
animals from the intermediately contaminated site
(Everglade Conservation Area 3A) exhibited elevated
thyroxine (T4 ) concentrations compared to animals from
both Belle Glade and West. Crain et al. (1998) found
elevated T4 concentrations in alligators taken from the
North shore of Lake Okeechobee, similar to that found
in animals from the Conservation Area 3A.
Both histological and hormonal data have been used
to indicate thyroid endocrine disruption. Recently, it has
been suggested that multiple indices are needed to accurately characterize such disruptions (Capen, 1998;
O’Connor et al., 1999). Considering histological endpoints, epithelial cell width and percent colloid are useful measurements with which to assess thyroid function.
Epithelial cell shape, which is indicative of thyroid activity, can be described by epithelial cell width. Columnar-shaped epithelial cells, characterized by small cell
widths, are usually found in follicles that have been actively stimulated by TSH. An unstimulated follicle tends
to have cuboidal-shaped epithelial cells, characterized
by large cell widths (Leatherland, 1994). The amount of
colloid present in a follicle is also an indicator of thyroid
function, with less colloid indicating hormonal secretion
from the gland (Moccia et al., 1981). Considering hormonal endpoints, T4 concentration is often used to assess disruption, as it is the major circulating thyroid
hormone (McNabb and King, 1993).
Recent studies of thyroid disruption have shown
concurrent alterations in histological and hormonal
parameters. Zhou et al. (1999) found that mummichogs
(Fundulus heteroclitus) exposed to heavy metals and
organic materials had larger thyroid follicles, greater
epithelial cell heights, and elevated circulating T4 concentrations. Similarly, rats exposed to low levels of
acrylamide exhibit decreased colloid area with a corresponding increase in plasma T4 (Khan et al., 1999).
However, the current study found that animals from the
most contaminated site (Belle Glade) had significantly
reduced colloid but apparently normal thyroxine. Additionally, animals from the moderately contaminated
site (Conservation Area) had normal histological parameters but significantly elevated plasma T4 . It is expected that as animals decrease their colloid (i.e., secrete
thyroid hormones into the blood), the plasma T4 will
rise. However, this is obviously not the case in animals
of both Belle Glade and Conservation Area, and there
are two possibilities for the discrepancy between tissue
and blood parameters.
First, there could be tissue-level thyroid disruption,
but the discrepancy could be a factor of the one-time
sampling event. The results of this study indicate that
Belle Glade animals, compared to those from the reference site, have either slightly flattened or cuboidalshaped epithelial cells coupled with a depleted source of
colloid, suggesting that thyroid hormone secretion has
occurred without concurrent thyroid hormone production (hormonogenesis). Similar abnormalities in colloid
content have been noted in salmon and herring gulls
from the Great Lakes (Moccia et al., 1981, 1986). Follicles largely depleted of colloid were common to both
species; however, Moccia et al. found epithelial cells to
be columnar in shape, unlike the cuboidal-shaped epithelial cells of alligators in this study. If thyroid-level
disruption was occurring in the alligators, we would
assume that the Belle Glade animals would have eventually had reduced T4 (corresponding with their reduced
colloid) and that previous to the sampling, Conservation
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E.A. Hewitt et al. / Chemosphere 47 (2002) 1129–1135
Area animals had reduced colloid. This is supported by
a recent study that found distinct phases of thyroid activity (hormonogenesis and hormone release), with a
lack of concurrent association between thyroid hormone
and colloid appearance (Raine and Leatherland, 1999).
Second, there may be thyroid endocrine disruption
both at the thyroid and at a site peripheral to the thyroid. Some xenobiotics have been show to disrupt thyroid economy by inducing hepatic microsomal enzymes
(Capen, 1997). Elevating such hepatic enzymes would
cause increased urinary excretion of thyroid hormones,
and this would explain the results from the Belle Glade
animals; whereas T4 production histologically appears
to be elevated (i.e., reduced colloid), the circulating T4
concentration is normal. It has been suggested that decreased T4 concentrations in male rats exposed to PCBs
result from increased hepatic metabolism of T4 by the
liver (Gray et al., 1993). If organochlorines are elevating
microsomal hepatic enzymes at the same time that T4
production is elevated, we would expect to see reduced
colloid coupled with ‘‘normal’’ T4 levels. This hypothesis
of thyroid disruption peripheral to the thyroid is supported by the abundance of organochlorine contaminants at the Belle Glade site; organochlorines are known
to induce hepatic microsomal enzymes (Capen, 1997).
Of these two possibilities, we support the latter because a combination of both thyroid gland and hepatic
disruption fully explains our data. Using West as our
reference site, the intermediately contaminated Conservation Area shows elevated circulating T4 , suggesting an
increase in hormone secretion. Table 1 shows that contaminants in animals from Conservation Area are similar to those from Belle Glade with the exception of
elevated organochlorines in the Belle Glade alligators.
Therefore, animals from Belle Glade appear to have
both elevated hormonal secretion and urinary excretion
of T4 .
The results in the present study suggest that abnormalities in thyroid histology and circulating thyroid
hormone concentrations exist in juvenile alligators on
Lake Okeechobee, specifically in animals collected from
sites of historical environmental contamination. Thus,
thyroid gland disruption, structural and functional, in
alligators appears to have an environmental etiology. In
order to obtain a more accurate indication of thyroid
activity, future research should include sampling at
multiple points throughout the year and utilize responses at the level of both the thyroid and peripheral to
the thyroid.
Acknowledgements
Advice on thyroid histology and assistance in histological procedures from Matthew Milnes, Brian Whitten, and Robert Roberts was greatly appreciated. We
also thank Matthew Milnes, Allan Woodward, and
Jenny Gates, among others, for assisting in obtaining
alligators used in this study. Fieldwork was conducted
under permit from the Florida Game and Fresh Water
Fish Commission. Funding for this research was provided by Environmental Protection Agency grants
CR826357-01-1 and CR162460-91-6.
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Elizabeth A. Hewitt graduated in May 2000 from Maryville
College. She graduated Magna Cum Laude with a B.A. in
Biology and a minor in Chemistry. This paper is the result
of Hewitt’s 1999 summer research experience at the Department
of Zoology, University of Florida.
D. Andrew Crain is currently Assistant Professor of Biology at
Maryville College. He graduated with a Ph.D. from the University of Florida in 1997. Dr. Crain has 25 publications exploring normal and contaminant-altered endocrine systems. In
2000, he and Dr Louis Guillette co-edited a book entitled
Environmental Endocrine Disruptors: An Evolutionary Perspective.
Mark P. Gunderson is a graduate student of Dr Lou Guillette’s
in the Department of Zoology, University of Florida. Mark
graduated Magna Cum Laude during the spring of 1995 from
St. Olaf College in Northfield Minnesota and was inducted into
the Phi Beta Kappa honor society that same spring.
Louis J. Guillette, Jr. is Professor of Zoology and Alumni
Distinguished Professor at the University of Florida. Dr Guillette received his doctorate in Comparative Reproductive Biology from the University of Colorado at Boulder in 1981. He
has served as a science advisor to many US and foreign agencies
regarding the developmental impacts of environmental contamination.
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